U.S. patent application number 11/518099 was filed with the patent office on 2007-04-26 for cochlear implants containing biological cells and uses thereof.
Invention is credited to Albert Edge, Stefan Heller.
Application Number | 20070093878 11/518099 |
Document ID | / |
Family ID | 37836534 |
Filed Date | 2007-04-26 |
United States Patent
Application |
20070093878 |
Kind Code |
A1 |
Edge; Albert ; et
al. |
April 26, 2007 |
Cochlear implants containing biological cells and uses thereof
Abstract
This invention generally relates to cochlear implants containing
or supporting stem-cell derived neural cells, and methods of making
and using the cochlear implants.
Inventors: |
Edge; Albert; (Newton,
MA) ; Heller; Stefan; (Menlo Park, CA) |
Correspondence
Address: |
FISH & RICHARDSON PC
P.O. BOX 1022
MINNEAPOLIS
MN
55440-1022
US
|
Family ID: |
37836534 |
Appl. No.: |
11/518099 |
Filed: |
September 8, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60715080 |
Sep 8, 2005 |
|
|
|
Current U.S.
Class: |
607/57 |
Current CPC
Class: |
A61N 1/0541 20130101;
A61N 1/36038 20170801; A61L 27/3834 20130101; A61L 27/3633
20130101; A61N 1/36121 20130101; A61L 2430/14 20130101; A61L 27/54
20130101; A61L 2300/414 20130101; A61N 1/326 20130101 |
Class at
Publication: |
607/057 |
International
Class: |
A61N 1/00 20060101
A61N001/00 |
Claims
1. A modified cochlear implant comprising a biological cell that is
operably associated with an electrode within the implant.
2. The modified cochlear implant of claim 1, wherein the biological
cell is a stem cell, a neural progenitor cell, or a neuron.
3. The modified cochlear implant of claim 2, wherein the stem cell
is an adult stem cell or an embryonic stem cell.
4. The modified cochlear implant of claim 1, further comprising a
holding area comprising additional biological cells.
5. The modified cochlear implant of claim 4, wherein the additional
biological cells comprise stem cells, neural progenitor cells, or
neurons.
6. The modified cochlear implant of claim 1, wherein the biological
cell or the electrode with which it is operably associated is at
least partially covered by a composition comprising a component of
the extracellular matrix (ECM).
7. The modified cochlear implant of claim 6, wherein the component
of the ECM is fibronectin, laminin, collagen, elastin, vitronectin,
a proteoglycan, or a glycosaminoglycan.
8. The modified cochlear implant of claim 1, wherein the biological
cell or the electrode with which it is operably associated is at
least partially covered by a composition comprising a cytokine.
9. The modified cochlear implant of claim 8, wherein the cytokine
is growth factor or chemokine that improves the survival, motility,
or differentiation of the biological cell.
10. The modified cochlear implant of claim 9, wherein the growth
factor is nerve growth factor (NGF), brain-derived neurotrophic
factor (BDNF), neurotrophin-3 (NT-3), ciliary neurotrophic factor
(CNTF), neurotrophin-4/5 (NT-4/5), glial-cell line derived
neurotrophic factor, leukemia inhibitory factor (LIF), or a
fibroblast growth factor (FGF).
11. The modified cochlear implant of claim 1, wherein the
biological cell or the electrode with which it is operably
associated is at least partially covered by hydrogel or a
composition comprising a polymer.
12. The modified cochlear implant of claim 11, wherein the hydrogel
is MATRIGEL.TM. or ECM GEL.
13. A method of treating a human patient who has experienced
hearing loss, the method comprising implanting the modified
cochlear implant of claim 1 in the human.
14. The method of claim 13, wherein the hearing loss is not
significantly improved by the use of an external hearing aid.
15. The method of claim 13, wherein the hearing loss is severe or
profound.
16. The method of claim 13, wherein the human patient is a child
between the ages of about 12 months and 48 months.
17. The method of claim 13, wherein the human patient is an elderly
patient.
18. The method of claim 13, wherein the method further comprises,
prior to implanting the modified cochlear implant, the step of
identifying a human patient in need of treatment.
19. The method of claim 13, wherein the method further comprises
subjecting the human patient to a rehabilitation regimen to
facilitate hearing, speech, and language skills.
20. The method of claim 13, wherein the method further comprises
administering to the human patient a pharmaceutical composition
comprising an active ingredient that serves as an analgesic,
antibiotic, or anti-inflammatory agent.
21. The method of claim 13, wherein the method further comprises
administering to the human patient a stem cell, a neural progenitor
cell or a neuron, wherein the stem cell, neural progenitor cell or
neuron is administered to the ear containing the modified cochlear
implant, and the stem cell, neural progenitor cell or neuron is not
associated with the modified cochlear implant.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/715,080, filed Sep. 8, 2005, which is
incorporated herein by reference in its entirety.
TECHNICAL FIELD
[0002] This invention generally relates to cochlear implants
containing biological cells and methods of making and using the
cochlear implants.
BACKGROUND
[0003] A cochlear implant is an electronic device that is implanted
into the inner ear to restore auditory perception, at least
partially, to the deaf and hard-of-hearing. Cochlear implants
create auditory sensation by generating electric field gradients in
the area of the peripheral nerve fibers of the auditory nerve
bundle. This bundle contains approximately 30,000 individual
afferent nerve fibers normally linked to approximately 4,500 inner
hair cells. Sound signals are picked up by a microphone within the
implant, converted into digital signals, and processed by a signal
processor in order to activate different stimulation channels.
These channels, in turn, stimulate different groups of nerve fibers
within the auditory nerve.
[0004] The ear is composed of four main sections: the external ear,
middle ear, inner ear, and the transmission pathway to the hearing
center in the brain. In normal hearing, sound waves travel along
the external ear canal and cause the tympanic membrane (also called
the ear drum) to vibrate. The three small bones of the middle ear
(the malleus, incus, and stapes) transmit these vibrations to the
cochlea of the inner ear. The cochlea is divided along its length
by a basilar membrane that distributes vibrational energy
longitudinally by frequency. The lowest frequencies cause maximum
membrane motion near the cochlea's apex, and the highest
frequencies maximize motion near the base. Four parallel rows of
hair cells extend along the length of the basilar membrane and,
when vibrated, transduce acoustic signals into electrical impulses
carried to the brain by auditory nerve fibers (see FIGS. 1 and
2).
SUMMARY
[0005] The present invention features devices for treating hearing
loss, methods of making the devices, and methods for treating
hearing loss. The devices include cochlear implants with biological
cells placed at least on one or more of the electrodes of the
implant. While cochlear implants are described further below, we
note here that they include electrodes that bypass dead or damaged
hair cells in the cochlea by directly stimulating the auditory
nerve fibers leading to the perceptions of sound. The cells can
extend processes from the implant to the brainstem, creating a
bridge that transmits electric signals from the electrode to the
brainstem more efficiently than traditional cochlear implants.
Methods of making the implants include applying a composition
containing biological cells to an electrode of the implant, and
treatment methods include implanting a cochlear implant that
carries cells on one or more electrodes into a human who has
experienced hearing loss (e.g., sensorineural hearing loss).
[0006] In one aspect, the invention features a cochlear implant
that carries a biological cell that is operably associated with an
electrode within the implant. The biological cell can be, for
example, a stem cell, a neural progenitor cell, or a neuron. The
neural progenitor cell or neuron can be derived from a stem cell or
from a more differentiated progenitor cell. The biological cell can
be embedded in a composition that covers the electrodes, fully or
partially. A neuron can be an afferent neuron (i.e., a neuron
having neural processes that extend towards the central nervous
system). For example, processes from an afferent neuron can extend
toward the brainstem and synapse with neurons there. In addition
to, or instead of, mature neurons, the composition can also include
stem cells or stem-cell derived neural progenitor cells, which are
not completely differentiated, but which express some genes that
are typically or exclusively expressed in neurons. The stem cells
are pluripotent, undifferentiated, and capable of differentiating
into a variety of different cell types. As these cells
differentiate into neurons, they can supplement any previously
present neurons and may therefore replace neurons if they
degenerate within the implant. The composition in which the cells
(e.g., stem cells, progenitor neural cells and differentiated
neural cells) are contained can include a component of the
extracellular matrix (ECM), including one or more of fibronectin,
laminin, collagen, elastin, vitronectin, a proteoglycan, a
glycosaminoglycan, and the like. The composition in contact with
the electrodes, or otherwise positioned with the device to
transduce acoustic signals, can also include a neurotrophin, such
as nerve growth factor (NGF), brain-derived neurotrophic factor
(BDNF), neurotrophin-3 (NT-3), ciliary neurotrophic factor (CNTF),
neurotrophin-4/5 (NT-4/5), glial-cell line derived neurotrophic
factor (GDNF), leukemia inhibitory factor (LIF), or fibroblast
growth factor (FGF). More generally, the neurotrophin can be any
compound or substance that stimulates neural cell survival or
differentiation, which may manifest as process outgrowth and which
may be mediated by the interaction with cellular receptors (e.g.,
tyrosine receptor kinase C (trkC), tyrosine receptor kinase B
(trkB)).
[0007] A cochlear implant carrying biological cells on its
electrodes can also have a separate holding area containing stem
cells or progenitor neural cells located near the electrodes. These
cells can differentiate to replace the degenerated neurons of the
human implantee or to replace neural cells (including neurons and
progenitor neural cells) of the implant, should they degenerate.
This store of cells can also include undifferentiated stem cells
and neurons (e.g., afferent neurons). The holding area can consist
of one or more dimples or grooves on the implant, such as on the
region of the implant between the implanted stimulator/receiver
unit and the electrode bundle, and nearer the electrode bundle.
[0008] In another aspect, methods of making a cochlear implant are
provided. These methods include the step of applying a cell (e.g.,
a stem cell, a more differentiated stem cell-derived progenitor, or
a recognizable neural cell) to one or more of the structures within
the implant (e.g., the electrodes of the cochlear implant). The
composition applied can further include a component of the ECM (as
noted above, suitable components include fibronectin, laminin,
collagen, elastin, vitronectin, a proteoglycan, a
glycosaminoglycan, and the like). The composition can also include
a neurotrophin, such as one or more of NGF, BDNF, NT-3, CNTF,
NT-4/5, GDNF, LIF, and FGF. The cells applied to the implant can be
associated with a hydrogel or other porous matrix. Suitable and
commercially available hydrogels include MATRIGEL.TM. (BD
Biosciences, San Jose, Calif.) and ECM GEL (Sigma, St. Louis, Mo.).
A hydrogel is a substance formed when an organic polymer, which can
be natural or synthetic, is set or solidified to create a
three-dimensional open-lattice structure that entraps molecules of
water or other solutions to form a gel. Solidification can occur by
aggregation, coagulation, hydrophobic interactions, cross-linking,
or similar means. While the properties of the hydrogel or other
porous matrix can vary, they will typically be of a uniform density
and, therefore, allow a uniform distribution of cells. The hydrogel
or matrix can contain varying numbers of cells (e.g., about 1-100
million cells per ml). The nature of the hydrogel or matrix allows
diffusion of nutrients, neurotrophins, cellular metabolites, and
the like, to and from the implant and thereby facilitates the
survival and/or differentiation of the cells within. Where a
hydrogel is used, it can include one or more of a polysaccharide, a
protein, a polyphosphazene, a poly(oxyethylene)-poly(oxypropylene)
block polymer, a poly(oxyethylene)-poly(oxypropylene) block polymer
of ethylene diamine, a polyacrylic acid, a poly(methacrylic acid) a
copolymer of acrylic acid and methacrylic acid, poly(vinyl
acetate), or a sulfonated polymer. The composition can
alternatively be a porous matrix that is not a hydrogel. The porous
matrix can be, or can include, a sponge, a foam, a calcium
carbonate matrix (e.g., coral or hydroxyapatite), or a rigid
inorganic ceramic, or metal structure having internal pores (e.g.,
a honeycomb). For example, the matrix can be made from titanium, or
can be a skeleton or mesh of fine struts and/or thick struts. The
fine struts can include thin interwoven polymer fibers and the
thick struts can be a network of metal, inorganic, ceramic, or
plastic rods. Where a polymer is used, it can be a polymer of
polylactic acid (PLA), polyglycolic acid (PGA), or a copolymer
thereof (PLGA). We may refer to the hydrogel and the other porous
matrices as support structures.
[0009] ECM components and growth factors, such as a neurotrophin
(e.g., NT-3) can be included in the device and may be embedded in
the matrix or immobilized on the matrix. The porous matrix can
support cell chemotaxis. A hydrogel or porous matrix used with a
cochlear implant is biocompatible (e.g., non-toxic to cells).
[0010] An optional step in making a cochlear implant featured in
the invention includes applying a separate concentrated store of
stem-cell-derived neural progenitor cells or neurons near the
electrodes, such as in dimples or grooves etched into the implant.
This holding area may also be a lumen or another attached reservoir
for storing the cells. The cells in this separate holding area can
be embedded in a composition including extracellular matrix
proteins, and optionally, neurotrophins, as described above.
Undifferentiated stem cells, and differentiated neurons can also be
stored in this separate holding area.
[0011] One aspect featured in the invention includes a modified
cochlear implant for use in the treatment of a hearing loss. The
modified cochlear implant can include any implant described herein,
including implants having biological cells placed at least on one
or more electrodes of the implant.
[0012] In one aspect, the invention features a method of treating a
human by implanting a cochlear implant carrying cells into the
human. The cochlear implant can be implanted into the scala tympani
of the ear, or the device can be implanted outside of the cochlea,
such as in the middle ear, or in the inner ear, and near the
auditory nerve. The treatment methods can include a step of
identifying a human in need of treatment, such as identifying a
human whose hearing does not improve following use of an external
hearing aid. A human who is a candidate for treatment with a
cochlear implant carrying cells can have unilateral or bilateral
hearing loss, and/or severe or profound hearing loss. The human can
be a child, typically having an age of from between about 12 months
and 18 years, or the human can be an adult over the age of 18
years. Following treatment with a cochlear implant featured in the
invention, the human can follow a rehabilitation regimen, where the
implantee builds skills, including hearing, speech and language
skills. The rehabilitation regimen can be maintained for 6 months,
one year, or longer than a year. The human can attend
rehabilitation sessions more frequently in the months immediately
following implantation of the device and less frequently as skill
levels become more advanced.
[0013] The treatment methods featured in the invention can include
implanting biological cells into the inner ear of the human, in
addition to the implantation of the modified cochlear implant,
which has a composition on the electrodes that includes biological
cells. These supplemental biological cells implanted into the inner
ear of the modified cochlear implant recipient can be any of the
biological cells described above, which are also suitable for use
on the modified cochlear implant. For example, the supplemental
implanted cells can include stem cells, neural progenitor cells, or
neurons, or a combination of these cell-types. Supplemental
progenitor cells or neurons can be derived from a stem cell or from
a more differentiated progenitor cell. The supplemental biological
cells can be implanted into the inner ear, such as near the base of
the auditory nerve near its connection with the cochlea. The cells
can alternatively be implanted into the modiolus of the cochlea.
The supplemental cells can extend processes towards the auditory
nerve, as well as towards the cochlear implant to provide
additional connections between the implant and the auditory nerves,
thereby providing additional neural connections which can further
increase the efficiency of the transmission of electric signals
from the electrodes of the implant to the brainstem. There may be
certain advantages to the use of cochlear implants that include
cells. For example, traditional cochlear implants (i.e., implants
that do not include cells) do not form connections with the
auditory nerve, and so the electrical stimulation emanating from
the implant must travel through extracellular fluid, connective
tissue and myelinated tissue to reach the auditory nerve cells. The
distance the electrical stimulation has to travel to reach the
auditory nerve cells and the electrical resistance of these
obstacles weakens the effect of the electrical stimulation.
Providing neural cells to the implant, however, can generate a
bridge between the implant and the auditory nerves (e.g., the
spiral ganglion neurons) as the processes of the neural cells grow
outwards to form synaptic connections with the auditory nerve.
Alternatively, the neural cells of the implant can functionally
replace the auditory neurons (e.g., the spiral ganglion
neurons).
[0014] Other features and advantages of the invention will be
apparent from the accompanying description and the claims. The
contents of all references, pending patent applications and
published patents, cited throughout this application are hereby
expressly incorporated by reference. In case of conflict, the
present specification, including definitions, will control.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 is a diagram illustrating a cochlear implant (shown
in black) positioned within a patient, bypassing the normal
pathways of the ear canal and the three bones of the middle ear
(Eddington and Pierschalla, "Cochlear Implants: Restoring Hearing
to the Deaf," On the Brain (The Harvard Mahoney Neuroscience
Institute Letter) Vol. 3, No. 4, Fall 1994).
[0016] FIG. 2 is a diagram of an implanted cochlea. The electrodes
are positioned to activate the auditory nerve fibers (Eddington and
Pierschalla, "Cochlear Implants: Restoring Hearing to the Deaf," On
the Brain (The Harvard Mahoney Neuroscience Institute Letter) Vol.
3, No. 4, Fall 1994).
[0017] FIG. 3A is a graph illustrating the compound action
potential (CAP) threshold elevation in de-afferented and control
cat ears. The auditory nerve was cut 10 weeks prior to taking the
measurements.
[0018] FIG. 3B is a graph illustrating the distortion product
otoacoustic emissions (DPOAEs) in the de-afferented and control cat
ears. The auditory nerve was cut 10 weeks prior to taking these
measurements.
DETAILED DESCRIPTION
[0019] We now further describe devices and methods for treating
deafness and hearing loss in a human. The device can be a cochlear
implant that has been modified by inclusion of cells, and the
methods include implantation of such a device.
[0020] Cochlear Implants. A cochlear implant is an electronic
device that is used to improve hearing in humans who have
experienced hearing loss, particularly severe to profound hearing
loss. These devices typically include an "external" and an
"internal" part. The external part includes a microphone, which can
be placed behind the ear, that detects sounds in the environment.
The sounds are then digitized and processed by a small computer
called a speech processor. The external components may be referred
to as a processor unit. In addition to the microphone and speech
processor, the external portion of the implant can include a power
source, such as a battery and an external antenna transmitter coil.
The internal part is an electronic device that is put under the
skin in the vicinity of the ear and is commonly referred to as a
stimulator/receiver unit (see FIG. 1). The coded signal output by
the speech processor is transmitted transcutaneously to the
implanted stimulator/receiver unit situated within a recess of the
temporal bone of the implantee. This transcutaneous transmission
occurs through use of an inductive coupling provided between the
external antenna transmitter coil which is positioned to
communicate with the implanted antenna receiver coil provided with
the stimulator/receiver unit. The communication is typically
provided by a radio frequency (RF) link, but other such links have
been proposed and implemented with varying degrees of success.
[0021] The implanted stimulator/receiver unit typically includes
the antenna receiver coil that receives the coded signal and power
from the external processor component, and a stimulator that
processes the coded signal and outputs a stimulation signal to an
electrode assembly, which applies the electrical stimulation
directly to the auditory nerve producing a hearing sensation
corresponding to the original detected sound.
[0022] An electrode connected to the electronic device is inserted
into the inner ear. The electrode can be a bundle of wires that
have open contacts spread along the length of the cochlea and
represent different frequencies of sounds. The number of electrodes
can vary from 1 to about 30 electrodes, such as about 5, 10, 15,
18, 20, 22, 24, 26, or 28 electrodes.
[0023] The biological cells described herein can be applied to any
cochlear implant, regardless of the number of electrodes employed,
the size of the speech processor, or other variations. The external
components of the cochlear implant can be carried on the body of
the implantee, such as in a pocket of the implantee's clothing, a
belt pouch or in a harness, while the microphone can be attached to
a clip and mounted behind the ear or attached to a piece of
clothing, such as a lapel. Alternatively, the external components
can be housed in a smaller unit capable of being worn behind the
ear. Such a unit can house the microphone, power unit and the
speech processor. In yet another alternative, the components that
are traditionally housed externally may be reduced in size to be
implanted or worn in the ear (or a combination of both), resembling
a hearing aid. For example, the battery and microphone may be
housed in a unit worn in the ear, while the speech processor is
implanted behind the ear.
[0024] Functionally, cochlear implants attempt to mimic the hair
cells of the cochlea. Hair cells are the sensory cells that convert
sound-derived mechanical stimulation into electrical signals that
are relayed to the brain via auditory ganglion nerve cells (also
called auditory neurons). Like hair cells, cochlear implants
stimulate auditory neurons. This stimulation is typically achieved
by surgically introducing the electrodes of the cochlear implant
into the scala tympani of the cochlear spiral. An electronic device
connected to the electrodes converts sound into impulses of
different frequencies that stimulate the auditory neurons. The
electrical stimulation has to travel through extracellular fluid,
connective tissue and myelinated tissue to reach the auditory
neurons, and the resistance created by these obstacles decreases
the effect of the electrical stimulation. The inclusion of cells
within the implant, however, as described herein, can create a
direct physical connection between the implant and the patient's
nervous system as the processes of the neural cells grow outwards
to form synaptic connections with the auditory nerves, therefore
enhancing stimulation of the auditory nerve, or to functionally
replace one or more auditory neurons.
[0025] The biological cells can be operably associated with (e.g.,
attached to) a surface of the implant. A biological cell is
operably associated with a cochlear implant when the cell,
following implantation into a patient, transmits an electrical
signal from the implant (e.g., from an electrode within the
implant) to a cell within the patient's nervous system (e.g., to a
cell within the auditory nerve) and thereby improves the patient's
hearing. For example, the cells can be attached to a surface of the
electrode bundle or to one or more of the electrodes themselves. In
the latter case, neurites will extend from the cells through
openings in any casing that may surround the electrode bundle. As
described further below, the cells can be applied in a composition
that may include one or more of a tissue culture medium (some of
which may remain associated with the cells as they are moved from a
tissue culture or storage vial), a polymer or gel, or a bio-active
compound such as ECM protein or a growth factor. Alternatively, or
in addition, one or more of these agents can be applied to the
outside of the electrode bundle, over the electrodes themselves, or
both as appropriate. The bioactive compounds support cell survival
and may promote neurite outgrowth. Accordingly, a bioactive
compound is one that upon inclusion in a modified cochlear implant,
improves cell survival and/or neurite outgrowth relative to what
would occur in the compound's absence.
[0026] The modified cochlear implant described herein can be
implanted in any way traditional, unmodified implants are implanted
(e.g., into the scala tympani). Alternatively, the modified implant
can be positioned outside the cochlea, but in close vicinity to the
auditory nerve (e.g., near enough that cells associated with the
modified implant can form functional connections with the patient's
auditory nerve fibers or other parts of the auditory nervous
system). For example, if the auditory nerve is degenerated (e.g.,
due to trauma or genetic disorder), the implant can be positioned
near the pathway of the lost auditory nerve fibers.
[0027] More specifically, cells attached to the implant will grow
processes having the signal-transmitting properties of neurites.
The processes will extend from the matrix coating the electrode
(where applied or when present), or other area of the implant, and
synapse with fibers within the auditory nerve. Alternatively, or in
addition, the processes can extend along the pathway where the
auditory nerve was located before fibers within it degenerated, and
into the auditory brainstem. The neurites can synapse with
endogenous auditory nerve fibers or with auditory neuron cell
bodies. Neurites that extend to the auditory brain stem can connect
with appropriate target cells in a way that sound perception is at
least partially restored. Target cells, for example, include cells
of the cochlear nucleus and other neurons along the central
auditory pathways. For example, the neurites can connect with
postsynaptic neurons in the cochlear nucleus, or can extend along
the central auditory pathways to connect with neurons in the
superior olivary nuclei; the nuclei of the lateral lemniscus,
inferior colliculus, or medical geniculate nucleus; or the neurites
can extend into the auditory cortex. The cell bodies that are
initially positioned on the modified cochlear implant may remain on
the implant or may migrate along the nerve fibers to form a
structure that functionally replaces or supplements the auditory
ganglion.
[0028] As described further below, the modified cochlear implant
can include one or more cell types, including stem cells, neural
cell progenitor cells, which may be derived from stem cells, and
mature neurons. Progenitor cells are cells that are not fully
differentiated. They can be derived from stem cells, which are
pluripotent, and can be induced to differentiate into a variety of
cell types. Stem cells or progenitor cells can be positioned within
the modified implant (e.g., along the electrodes) with
differentiated neural cells, and the stem cells or progenitor cells
may also be included in a holding area in close vicinity to the
electrodes. This store will replace cells that have degenerated in
the ear and will also supplement the neural cells that form
processes that synapse with auditory neurons and the auditory
brainstem. Some of the stem cell-derived progenitor neural cells
may remain in the modified cochlear implant, and some of the
progenitor cells may migrate out of the device, either remaining
quiescent or differentiating into additional neurons that connect
the implant's electrodes with either the brainstem or with the
remaining auditory neurons.
[0029] The modified cochlear implants can include a polymer, which
may be within a hydrogel, or another porous substance.
Extracellular matrix (ECM) proteins can be included in the
composition, which may contain fibronectin and laminin, as well as
collagens, proteoglycans, glycosaminoglycans, elastin, vitronectin
and the like. The extracellular matrix component can include a
matrigel, which is composed of collagen type IV, laminin, and
heparan sulphate. The gelation of an ECM component can be carried
out in the usual manner. For example, if the ECM component is
collagen, then a collagen gel can be obtained by incubating an
aqueous solution of 0.3-0.5% collagen at 37.degree. C. for 10-20
minutes. If necessary, a gelling agent may be used in gelation of
an ECM component. The gel matrix can include gelatin or agarose or
any natural or synthetic extracellular proteinous matrix or basal
membrane mimic including, but not limited to MATRIGEL.TM. (BD
Biosciences, San Jose, Calif.), or ECM GEL, (Sigma, St. Louis,
Mo.), or other hydrogels containing certain factors such as
cytokines (e.g., growth factors and chemokines), antibodies, or
other ligands for cell surface receptors. Preferably, the gel has a
substantially high water content (e.g., water content of 60%, 65%,
70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99%) and is porous enough to allow cell chemotaxis. Alternatively,
the polymer can be a porous matrix exclusive of a hydrogel.
[0030] The gel or polymer mix can contain other factors that
support neural cell growth and survival such as one or more
neurotrophins. Neurotrophins are molecules that support the
survival of different classes of neurons and include NT-3, BDNF,
GDNF, NGF, CNTF, NT-4/5, LIF, and FGF. For example, neurotrophin
NT-3 can be present in the polymer or gel where it interacts with a
receptor, such as the trkC receptor, on the neural cells.
Activation of the trkC receptor can promote neural cell outgrowth
and survival. In another example, NT-4/5 can be present in the
polymer or gel where it interacts with a receptor, such as the trkB
receptor, to support cell growth and survival. Other molecules that
can agonize or antagonize the receptors, or act as mimics of
neurotrophins or other chemoattractant agents, can be included in
the polymer or gel. Agonists of the receptors can, for example,
provide trophic support to the co-grafted neurons. These can
include antibodies, peptides, nucleic acids, and small molecule
chemical compounds. For example, monoclonal antibody Mab2256 can be
included in a polymer or matrix to agonize the trkC receptor (Ruiz
et al., Hum. Mol. Genet. 14:1825-1837, 2005, E-published May 1,
2005). In another example, the small molecule beta-turn
peptidomimetic D3 can be included in a polymer or matrix to agonize
the trkA receptor (Maliartchouk et al., Mol. Pharmacol. 57:385-391,
2000). Other molecules such as adenosine or synthetic adenosine
agonists (e.g., CGS 21680) can act through other receptors such as
the adenosine 2A receptor to activate Trk receptors (Lee and Chao,
PNAS 98:3555-3560, 2001). This interaction provides trophic support
for the neural cells of the modified cochlear implant. Optionally,
therapeutic agents such as analgesics, antibiotics,
anti-inflammatories, steroids, and the like can also be included in
the polymer mix.
[0031] The ECM proteins, neurotrophins and other factors for
supporting cell growth can be immobilized on the polymer or gel and
can interact with the cell receptors to encourage outgrowth, or to
encourage differentiation of stem cells or stem cell-derived
progenitor cells.
[0032] The polymer or gel mix, including biological cells, and
optionally the ECM proteins and growth factors can be applied to an
implant (e.g., used to coat the electrode bundle of the implant).
Alternatively, the polymer or gel mix can be applied to the implant
(e.g., to the electrode bundle) and the cells applied in a second
step. Regardless of the order of application, the cells can be stem
cells, progenitor cells, mature neurons, or a combination
thereof.
[0033] The modified cochlear implant can also include one or more
lumens or reservoirs that contain bioactive compounds or
pharmaceutical compositions (e.g., to prevent bleeding, scarring)
that can be released over time. This extra store of factors may
facilitate an enhanced and enduring effect on neurite outgrowth,
prevent neuron degeneration, and may help to optimize the overall
health of the implant.
[0034] Cells. The neural cells and progenitor cells appropriate for
attachment to a modified cochlear implant can be derived from stem
cells. Stem cells are unspecialized cells capable of extensive
proliferation. Stem cells are pluripotent and are believed to have
the capacity to differentiate into most cell types in the body
(Pedersen, Scientif Am. 280:68, 1999), including neural cells,
muscle cells, blood cells, epithelial cells, skin cells, and cells
of the inner ear (e.g., sensory cells, such as hair cells, and
auditory nerve cells, such as spiral ganglion cells). Stem cells
are capable of ongoing proliferation in vitro without
differentiating. As they divide, they retain a normal karyotype,
and they retain the capacity to differentiate to produce adult cell
types. Stem cells can differentiate to varying degrees. For
example, stem cells can form cell aggregates called embryoid bodies
in hanging drop cultures. The embryoid bodies contain neural
progenitor cells that can be selected by their expression of an
early marker gene such as Sox1 and the nestin gene, which encodes
an intermediate filament protein (Lee et al., Nat. Biotech.
18:675-9, 2000).
[0035] Stem cells useful for generating neural cells for growth on
a cochlear implant can be derived from a mammal, such as from a
human, mouse, rat, pig, sheep, goat, or non-human primate. If cells
from a non-human mammal are applied to the modified cochlear
implant, then an immunosuppressant will also be administered to
prevent rejection. Exemplary immunosuppressants include
antiproliferative agents (e.g., azathioprine, cyclophosphamide, and
methotrexate); anti-inflammatory agents, such as corticosteroids
(e.g., prednisone and prednisolone); and inhibitors of lymphocyte
activation, such as cyclosporines (e.g., cyclosporine A,
cyclosporine B, and cyclosporine G) and FK506. Other suitable
immunosuppressive agents include statins, TGF.beta.1, uteroglobin,
TH2 cytokine, rapamycin, mycophenylate and antibodies against
adhesion molecules or T cell signaling molecules. For example,
antibodies against the T cell signaling molecules B7 and CD28 are
suitable immunosuppressive agents.
[0036] Stem cells can be derived from any number of tissues
including, but not limited to, ear, eye, bone marrow, blood, or
skin. For example, stem cells have been identified and isolated
from the mouse utricular macula (Li et al., Nature Medicine
9:1293-1299, 2003). Stem cells useful for generating neural cells
can be adult stem cells, and therefore derived from differentiated
tissue, or the cells can be from embryonic tissue (called
"embryonic stem cells" or "ES cells"). The stem cells can also be
derived from umbilical cord fluid.
[0037] Methods of culturing neurons and neural progenitor cells for
attachment to a cochlear implant include culturing stem cells under
conditions that cause a stem cell to differentiate into a neural
cell or progenitor neural cell (see below and, e.g., Lee et al.,
Nat. Biotechnol. 18:675-9, 2000). The cells derived from stem cells
and applied to the cochlear implant need not be fully
differentiated to be therapeutically useful. A partially
differentiated cell that can further differentiate into a neural
cell can be applied to the modified cochlear implants described
herein. The cells applied to the modified cochlear implant need
only to be differentiated enough, or have the capability to
differentiate to an extent that allows them to convey information
from the electrodes of the implant to the patient's auditory
nervous system.
[0038] Neurons or neural progenitor cells can be generated from
stem cells isolated from a human, such as from the intended
recipient of the modified cochlear implant, or from a matched
donor. A matched donor will have a compatible ABO blood type. A
donor will preferably have identical or nearly identical human
leukocyte antigens (HLAs) as the implant recipient (e.g., 6 of 6,
or 5 of 6, or 4 of 6 of the donor's HLA profile will match the
recipient's). Stem cells can be derived from embryonic tissue or
from umbilical cord fluid, or stem cells can be derived from mature
(e.g., adult) tissue, such as tissue from the inner ear, central
nervous system, blood, skin, eye, bone marrow, or other accessible
tissue. Any method for culturing stem cells and inducing
differentiation into neural cells can be used.
[0039] A stem cell or progenitor cell can be isolated from the
inner ear of an animal. These methods include providing tissue from
the inner ear of the animal, where the tissue includes at least a
portion of the utricular maculae. The animal can be a mammal, such
as a mouse, rat, pig, rabbit, goat, horse, cow, dog, cat, primate,
or human. The isolated tissue can be suspended in a neutral buffer,
such as phosphate buffered saline (PBS), and subsequently exposed
to a tissue-digesting enzyme (e.g., trypsin, leupeptin,
chymotrypsin, and the like) or a combination of such enzymes, or a
mechanical (e.g., physical) force, such as trituration, to break
the tissue into smaller pieces. In one alternative, both mechanisms
of tissue disruption are used. For example, the tissue can be
incubated in a solution containing about 0.001-1.0% enzyme (e.g.,
about 0.001%, 0.01%, 0.03%, 0.05%, 0.07%, or 1.0% of enzyme) for
about 5, 10, 15, 20, or 30 minutes, and following incubation, the
cells can be mechanically disrupted. The disrupted tissue can be
passed through a device, such as a filter or bore pipette, that
separates a stem cell or progenitor cell from a differentiated cell
or cellular debris. The separation of the cells can include the
passage of cells through a series of filters having progressively
smaller pore size. For example, the filter pore size can range from
about 80 .mu.m or less, about 70 .mu.m or less, about 60 .mu.m or
less, about 50 .mu.m or less, about 40 .mu.m or less, about 30
.mu.m or less, about 35 .mu.m or less, or about 20 .mu.m or less.
The cells can be frozen for future use or placed in culture for
expansion or differentiation. The cells can be maintained on a
feeder layer of cells. For example, embryonic stem cells can be
maintained on a feeder layer of mitotically inactivated primary
mouse embryonic fibroblasts, as described in Pirity et al. (Methods
Cell Biol. 57:279-93, 1998). Optionally, a cytokine, such as LIF
can be added to the maintenance medium.
[0040] The separated cells can be placed in individual wells of a
culture dish at a low dilution and cultured to differentiate into
cells having characteristics of cells of the inner ear or of neural
cells. The cells can be distributed in the culture dish so that
each dish, or a well therein, includes only a single cell. To
initiate in vitro differentiation of embryonic stem cells in
hanging drop cultures, the cells can be removed from the feeder
layer and LIF. This can stimulate the formation of embryoid bodies
in hanging drops. The cells can be allowed to differentiate to
various stages. Thus, partially or more fully differentiated neural
cells are useful for attachment to a cochlear implant. Formation of
spheres (clonal floating colonies) from the isolated cells can be
monitored, and the spheres can be amplified by disrupting them
(e.g., by physical means) to separate the cells. These cells can be
cultured again to form additional spheres. Further culturing of the
cells in the absence of or in lower amounts of growth factors will
induce the spheres (and the cells of the spheres) to differentiate
further into more highly developed cells of the inner ear.
[0041] Appropriate stem cell culture medium is described in the
art, such as in Li et al. (Nat. Med. 9:1293-1299, 2003). For
example, stem cells can be cultured in serum free DMEM/high-glucose
and F12 media (mixed 1:1), and supplemented with N2 and B27
solutions and growth factors. Growth factors such as EGF, IGF-1,
and bFGF have been demonstrated to augment sphere formation in
culture. In vitro, stem cells often show a distinct proliferation
potential for forming spheres. Thus, the identification and
isolation of spheres can aid in the process of isolating stem cells
from mature tissue for use in making fully or partially
differentiated neural cells. The growth medium for cultured stem
cells can contain one or more or any combination of growth factors,
provided that the stem cells do not differentiate. To induce the
cells (and the cells of the spheres) to differentiate, the medium
can be exchanged for medium lacking growth factors. For example,
the medium can be serum-free DMEM/high glucose and F12 media (mixed
1:1) supplemented with N2 and B27 solutions. Equivalent alternative
media and nutrients can also be used. Culture conditions can be
optimized using methods known in the art.
[0042] The cells can be monitored for expression of cell-specific
markers. Neuronal progenitor cell populations can be identified by
expression of the marker protein nestin. Cells of the spiral
ganglion can be identified by the expression of ephrinB2, ephrinB3,
trkB, trkC, GATA3, BF1, FGF10, FGF3, CSP, GFAP, and Islet1.
Expression of cell-specific markers can be assayed by traditional
methods, including RT-PCR, Northern blot, Western blot, microarray
analysis (Lockhart et al., Nat Biotechnol 14:1675-1680, 1996), and
immunocytochemistry, which includes the staining of cells or
tissues using an antibody against a specific antigen. Expression of
these markers can be monitored before applying the cell to an
implant. Differentiation into neural cell types can also be
monitored by assaying for the development of neural cell
morphology, an acquired negative resting potential, and the ability
to fire action potentials. Resting potential and the ability to
fire action potentials can be assayed by known methods, including
extracellular single unit voltage recording, intracellular voltage
recording, voltage clamping, patch clamping, voltage sensitive
dyes, and ion sensitive electrodes.
[0043] In certain embodiments, the cells within a modified cochlear
implant (e.g., cells on or in close proximity to the electrodes)
can be modified by the induction of an exogenous vector that
includes a nucleic acid sequence encoding polypeptide. For example,
neural progenitor cells can be transfected with a vector for
overexpressing an .alpha..sub.v.beta..sub.3 integrin. These cells
would be expected to have an enhanced ability to extend neurites as
this integrin has been shown to mediate neurite extension from
ganglion neurons on laminin substrates (Aletsee et al., Audiol.
Neurootol. 6:57-65, 2001). Neurite extension can also be enhanced
by the addition of neurotrophins (e.g., BDNF, NT3 and LIF)
(Gillespie et al., NeuroReport 12:275-279, 2001). A Sonic hedgehog
(Shh) polypeptide or polypeptide fragment (e.g., Shh-N), can also
be useful as an endogenous factor to enhance neurite extension. Shh
is a developmental modulator for the inner ear and a
chemoattractant for axons (Charron et al., Cell 113:11 23, 2003).
The nucleic acid sequence encoding the polypeptide can be under the
control of a promoter, such as a constitutive promoter (e.g., a CMV
or human U6 promoter).
[0044] The stem cells, neural progenitor cells or differentiated
neural cells can be added to the ECM or hydrogel coating before
applying the coating to the modified cochlear implant, or the ECM
or hydrogel coating can be applied to the implant and then the
cells applied to the coating. The cells can be applied as separated
cells, or cell aggregates, such as embryoid bodies.
[0045] Treatment methods. Treatment methods include implanting a
modified cochlear implant into a subject who has a hearing loss.
Use of the modified cochlear implant can improve the ability of the
subject to hear.
[0046] A human having a hearing disorder can be fitted with a
modified cochlear implant that includes cells. As noted, the cells
can be attached to one or more electrodes or placed within the
implant in close proximity to the electrodes. Upon successful
implantation, processes will extend from the implant (e.g., from
the electrodes) and will form synaptic connections with the
auditory nerve, thereby stimulating the auditory nerve. The
implants of the invention are not limited however, to those in
which the cells form any particular connection or combination of
connections. The cells within the implant may migrate or extend
processes in a variety of ways to convey signals received from the
implant or the external environment to the patient's nervous system
in such a way that the patient experiences an improvement in the
ability to hear.
[0047] Any human experiencing a hearing loss is a candidate
recipient of a modified cochlear implant. A human having a hearing
loss can hear less well than the average human being, or less well
than on a prior occasion (e.g., less well than in years past or
than before an injury). For example, hearing can be diminished by
at least 5, 10, 30, 50% or more. The human can have mild hearing
loss (e.g., difficulty hearing sounds below an intensity range of
about 20 dB to 40 dB), moderate hearing loss (e.g., difficulty
hearing sounds below an intensity range of about 40 dB to 60 dB),
severe hearing loss (e.g., difficulty hearing sounds below an
intensity range of about 60 dB to 80 dB) or profound hearing loss
(e.g., difficulty hearing sounds below an intensity range of about
80 dB or higher). The human can have sensorineural hearing loss,
which results from damage or malfunction of the sensory part (the
cochlea) or the neural part (the auditory nerve) of the ear, or
conductive hearing loss, which is caused by blockage or damage in
the outer and/or middle ear. Alternatively, the human can have
mixed hearing loss, which is caused by a deficit in both the
conductive pathway (in the outer or middle ear) and in the nerve
pathway (the inner ear). An example of a mixed hearing loss is a
conductive loss due to a middle-ear infection combined with a
sensorineural loss due to damage associated with aging. The human
can have unilateral or bilateral hearing loss (loss of hearing in
one or both ears, respectively). To merit the use of an implanted
device, the loss of hearing in the human is likely to be so severe
that a hearing aid does not improve hearing.
[0048] The devices, compositions, and methods described herein are
appropriate for the treatment of hearing disorders resulting from
sensorineural hair cell loss or auditory neuropathy. Humans with
sensorineural hair cell loss experience the degeneration of
cochlear hair cells, which frequently results in the loss of spiral
ganglion neurons in regions of hair cell loss. Such humans may also
experience loss of supporting cells in the organ of Corti, and
degeneration of the limbus, spiral ligament, and stria vascularis
in the temporal bone material. Humans with auditory neuropathy
experience a loss of cochlear sensory neurons while the hair cells
of the inner ear remain intact.
[0049] The subject can be deaf or have a hearing loss for any
reason or as a result of any type of event. For example, a human
can be deaf because of a genetic or congenital defect; for example,
a human identified as a candidate for treatment can have been deaf
since birth, or can be deaf or hard-of-hearing as a result of a
gradual loss of hearing due to a genetic or congenital defect. In
another example, a human identified as a candidate for treatment
can be deaf or hard-of-hearing as a result of a traumatic event,
such as a physical trauma to a structure of the ear, or a sudden
loud noise, or a prolonged exposure to loud noises. For example,
prolonged exposures to concert venues, airport runways, and
construction areas can cause inner ear damage and subsequent
hearing loss. A human can experience chemical-induced ototoxicity,
wherein ototoxins include therapeutic drugs including
antineoplastic or chemotherapeutic agents, salicylates, quinines,
and aminoglycoside antibiotics, contaminants in foods or
medicinals, and environmental or industrial pollutants. A human can
have a hearing disorder that results from aging or that is
associated with a disease or disorder such as Meniere's disease,
multiple sclerosis, a brain tumor or a stroke.
[0050] Candidate recipients of the modified cochlear implants
described herein can be any age including children (e.g., children
about 12 months through 18 years) and adults over 18 years of age.
The modified cochlear implants may be especially beneficial to
hearing--impaired children between the ages of two and three, as it
is around this age that language skills develop the fastest.
[0051] A modified cochlear implant including attached cells can be
implanted in a human by any method known in the art. For example,
implantation can be performed under general anesthesia. Generally,
an incision is made behind the ear to expose the mastoid bone,
which is then removed to allow identification of the facial nerve
and the cochlea. An opening is then created in the cochlea to allow
insertion of the electrode array including the attached cells. An
insertion tool, or stylet, may be used to facilitate placement of
the electrode array inside the cochlea. To secure the implanted
processor and to reduce the prominence of the processor on the side
of the head, a bony depression can be drilled in the designated
position of the internal processor (typically above and behind the
outer ear). A permanent suture, Gortex sheeting, or any of a
variety of other means known in the field of cochlear implantation
can be used to secure the device into position. The skin incision
can then be closed using sutures, such as absorbable sutures, and a
compression/protective head wrap can be applied for 1, 2, or 3 days
or more to allow time for the skin incision to heal. The initial
activation of the implant may be delayed from 2, 3, 4, 5, or 6
weeks or more after surgery, to allow more time for recovery. The
electrodes may be activated in small batches, such that activation
of all the electrodes will occur over a period of several days or
weeks. A period of rehabilitation may follow activation of the
electrodes to build hearing, speech and language skills.
Rehabilitation sessions may occur weekly, bimonthly, monthly or
periodically over the course of up to a year or more. The
rehabilitation regimen can be maintained for 5 months, 6 months, 8
months, one year, or longer.
[0052] Before and after implantation of the modified cochlear
implant, and throughout a course of rehabilitation, the human can
be tested for an improvement in hearing. Methods for measuring
hearing are well-known and include pure tone audiometry, air
conduction, and bone conduction tests. These exams measure the
limits of loudness (intensity) and pitch (frequency) that a human
can hear. Hearing tests in humans include behavioral observation
audiometry (for infants to seven months), visual reinforcement
orientation audiometry (for children 7 months to 3 years) and play
audiometry for children older than 3 years. Brainstem evoked
response audiometry (BERA or ABR) can also be performed. In BERA,
sounds are placed in the ear, and the brainstem's response is
recorded from electrodes taped to the patient's head.
Electrocochleography can be used to provide information about the
functioning of the cochlea and the first part of the nerve pathway
to the brain.
[0053] The efficacy of the treatment methods described herein can
be assayed by determining an improvement in the subject's ability
to hear. Alternatively, efficacy can be assayed by measuring
distortion product otoacoustic emissions (DPOAEs) or compound
action potential (CAP). A patient is successfully treated upon
experiencing any objective or subjective improvement in their
hearing.
[0054] Certain imaging techniques can be used to supplement the
hearing tests. These include angiography, and magnetic resonance
angiography (MRA) in particular, to produce images of the blood
vessels to the brain, and SPECT or PET scans, which produce images
of microscopic blood flow within the brain.
[0055] Supplemental Therapies. Treatment with a modified cochlear
implant can be supplemented with the administration of cells (e.g.,
stem cells, partially differentiated progenitor cells, or more
differentiated neural cells) that are not attached to the implant.
These cells can be generated by the methods described herein. For
example, the cells can be transplanted, (e.g., in the form of a
cell suspension) into the ear by injection. For example, the cells
can be injected or otherwise placed into the luminae of the
cochlea. Injection can be, for example, through the round window of
the ear or through the bony capsule surrounding the cochlea. The
cells can be injected through the round window into the auditory
nerve trunk in the internal auditory meatus or into the scala
tympani. The cells can be implanted near the junction between the
cochlea and the brainstem. Alternatively, or in addition, the cells
can be implanted within the cochlea, such as within the scala
tympani, within the scala vestibuli, within the scala media, within
the modiolus, or within Rosenthal's canal.
[0056] To improve the ability of the cell to engraft, the
supplemental biological cells can be modified prior to
differentiation. For example, the cells can be engineered to
overexpress one or more anti-apoptotic genes in the progenitor or
differentiated neural cells. The FAK tyrosine kinase or Akt genes
are candidate anti-apoptotic genes that can be useful for this
purpose; overexpression of FAK or Akt can prevent cell death in
spiral ganglion cells and encourage engraftment when transplanted
into another tissue, such as an explanted Organ of Corti (see for
example, Mangi et al., Nat. Med. 9:1195-1201, 2003).
[0057] Supplemental cells implanted into the ear of the implant
recipient can form neurites that extend towards the brainstem and
can also form neurites that extend towards the electrode of the
cochlear implant. Neurotrophic factors within the matrix applied to
the electrodes of the implant or in releasable form in a chamber in
the cochlear implant can encourage the extension of neurites
towards the electrodes. For example, neurotrophins such as BDNF,
NT3, and leukemia inhibitory factor (LIF) included in the matrix
applied to the electrodes of the cochlear implant can enhance the
extension of neurites from the supplemental implanted cells towards
the electrodes where they can synapse with cells of the modified
cochlear implant. Sonic hedgehog (Shh) polypeptides or polypeptide
fragments (e.g., the N-terminus of Shh, Shh-N) included in the
matrix of the modified cochlear implant can also stimulate the
extension of neurites towards the electrodes of the implant. Shh is
a developmental modulator for the inner ear and a chemoattractant
for axons (Charron et al., Cell 113:11-23, 2000).
[0058] Treatment methods can also include administration of a
composition to the ear to encourage neural cell outgrowth from the
cochlear implant or to inhibit degeneration of the
implant-associated neural cells. These compositions can be the same
as or unique from those described above and associated with the
implant. For example, pharmaceutical compositions can include one
or more factors to enhance neural cell survival, migration, or
process extension, such as neurotrophins (e.g., BDNF, NT3, or LIF),
or a chemoattractant such as an Shh polypeptide or polypeptide
fragment (e.g., Shh-N).
[0059] A supplemental composition (i.e., a composition administered
in connection with the modified implant but not physically
associated with it) can include a steroid, a molecule to promote
healing or inhibit bleeding, inflammation, or scar tissue
formation.
[0060] The compositions of the invention, whether combined with
cells and used in connection with the implant or administered
separately can contain a carrier, many of which are known to
skilled artisans. Carriers that can be used include buffers (for
example, citrate buffer, phosphate buffer, acetate buffer, and
bicarbonate buffer), amino acids, urea, alcohols, ascorbic acid,
phospholipids, polypeptides (for example, serum albumin), EDTA,
sodium chloride, liposomes, mannitol, sorbitol, and glycerol. Where
a composition is administered independently from the implant, it
can be administered by any route of administration. For example,
liquid solutions can be made for administration by injection into
the ear. Methods for making such formulations can be found in, for
example, Remington's Pharmaceutical Sciences, 18th Ed., Gennaro,
ed., Mack Publishing Co., Easton, Pa., 1990.
[0061] The pharmaceutical compositions and methods described herein
can be used independently or in combination with one another to
supplement treatment with a modified cochlear implant. That is,
subjects can be administered one or more of the pharmaceutical
compositions in temporally overlapping or non-overlapping regimens.
The subject can also be administered a solution or tissue
containing the differentiated neural cells or stem-cell derived
progenitor cells described above. When therapies overlap
temporally, the therapies may generally occur in any order and can
be simultaneous or interspersed. Administration of a pharmaceutical
composition in addition to treatment with a modified cochlear
implant is optional. Treatment of hearing loss may consist solely
of administration of a modified cochlear implant as described
herein.
[0062] The formulations and routes of administration can be
tailored to the specific hearing disorder being treated, and for
the specific human being treated. For example, the human can have
been deaf from birth due to a genetic or environmental event, or a
child or adult human can be losing hearing due to environmental
factors such as prolonged exposure to loud noises, or a human can
be experiencing a hearing loss due to aging. Therefore the human
can be any age (e.g., an infant or an elderly person), and
formulation and route of administration can be adjusted
accordingly. A subject can receive a dose of the agent once or
twice or more daily for one week, one month, six months, one year,
or more. The treatment can continue indefinitely, such as
throughout the lifetime of the human. Treatment can be administered
at regular or irregular intervals (once every other day or twice
per week), and the dosage and timing of the administration can be
adjusted throughout the course of the treatment. The dosage can
remain constant over the course of the treatment regimen, or it can
be decreased or increased over the course of the treatment.
[0063] Generally the dosage facilitates an intended purpose for
both prophylaxis and treatment without undesirable side effects,
such as toxicity, irritation or allergic response. Although
individual needs may vary, the determination of optimal ranges for
effective amounts of formulations is within the skill of the art.
Human doses can readily be extrapolated from animal studies (Katocs
et al., Chapter 27 In: Remington's Pharmaceutical Sciences, 18th
Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990).
Generally, the dosage required to provide an effective amount of a
formulation, which can be adjusted by one skilled in the art, will
vary depending on several factors, including the age, health,
physical condition, weight, type and extent of the disease or
disorder of the recipient, frequency of treatment, the nature of
concurrent therapy, if required, and the nature and scope of the
desired effect(s) (Nies et al., Chapter 3, In: Goodman &
Gilman's The Pharmacological Basis of Therapeutics, 9th Ed.,
Hardman et al., eds., McGraw-Hill, New York, N.Y., 1996).
[0064] A pharmaceutical composition as described herein can be
packaged and labeled for use in conjunction with a modified
cochlear implant for treatment of a hearing disorder.
[0065] Pharmaceutical compositions can be formulated in a
conventional manner using one or more physiologically acceptable
carriers or excipients. For example, a composition can be
formulated for administration by drops into the ear, insufflation
(such as into the ear), topical, or oral administration.
[0066] In another mode of administration, the composition can be
directly administered in situ to the cochlea of the inner ear, such
as via a catheter or pump. A catheter or pump can, for example,
direct a differentiation agent into the cochlear luminae or the
round window of the ear.
[0067] In another route of administration, a differentiation agent
can be injected into the ear, such as into the luminae of the
cochlea (e.g., the Scala media, Sc vestibulae, and Sc tympani).
Injection can be, for example, through the round window of the ear
or through the cochlear capsule.
[0068] The invention is further illustrated by the following
examples, which should not be construed as further limiting.
EXAMPLES
Example 1
Neurons were Isolated from the Inner Ear of a Pig Fetus for use in
Transplantation Studies
[0069] We isolated pig fetal spiral ganglion cells from the inner
ear after timed pregnancies and placed the cells in culture for
periods up to two weeks. Gestational ages of E36, E41, E49, E60 and
E63 were compared. Following isolation of whole cochlea, the
tissues containing spiral ganglion cells were separated from other
tissues and incubated with trypsin-EDTA at 37.degree. C. for 10
minutes. After three washes with PBS plus DNAse, tissues were
triturated with three pre-calibrated flame polished Pasteur
pipettes with progressively smaller apertures. Cells were
resuspended in PBS plus glucose solution at approximately
1.times.10.sup.8/ml. The viability of the cells was determined by
trypan blue exclusion assay prior to transplantation. Some cells
were plated on poly-D lysine-coated 12-well culture plates in
complete neurobasal medium.
[0070] Immunohistochemical staining revealed that the E36 neurons
did not express neurofilament but did express neuron specific
enolase. At days E49 and later, the neurons expressed neuron
specific enolase and neurofilament as well as galactocerebrosidase.
The later time points yielded an increased ratio of connective
tissue components relative to neurons. The best yield of cells was
at E41, and these cells could be stained with all three of the
markers. This time point was therefore selected for the isolation
of cells for transplantation.
Example 2
Embryonic Stem Cell Cultures were Established and Controlled
Differentiation of Different Cell Types was Observed
[0071] We established cultures of the murine ES cell lines
YC5/EYFP, a derivative of the totipotent cell line R1 (Nagy et al.,
Proc. Natl. Acad. Sci. USA 90:8424-8, 1993); ROSA26-6; and Sox1-GFP
(Aubert et al., Nat. Biotechnol. 20:1240-5, 2002). YC5/EYFP cells
carry the gene for enhanced yellow fluorescent protein (EYFP) under
control of a promoter composed of a cytomegalovirus (CMV) immediate
early enhancer coupled to the .beta.-actin promoter (Hadjantonakis
et al., Mech. Dev. 76:79-90, 1998). ROSA26-6 cells and their
derivatives express the lacZ gene encoding the bacterial
beta-galactosidase enzyme (Pirity et al., Methods Cell Biol.
57:279-93, 1998). The Sox1-GFP cells express GFP controlled by the
promoter for the early neural marker Sox1.
[0072] Low passage ES cells are maintained on a feeder layer of
mitotically inactivated primary mouse embryonic fibroblasts (Pirity
et al., Methods Cell Biol. 57:279-93, 1998). Undifferentiated ES
cells proliferate actively and form compact clusters of small
cells. We initiated in vitro differentiation of ES cells in hanging
drop cultures in the absence of embryonic fibroblast feeder cells
and of leukemia inhibitory factor (LIF), a cytokine that promotes
the pluripotency of ES cells. Within two days, cell aggregates of
uniform size termed embryoid bodies form in the hanging drops.
[0073] Using a published protocol (Lee et al., Nat. Biotechnol.
18:675-9, 2000), we were able to select neural progenitor cell
populations that express the defining marker protein nestin.
Nestin-positive progenitors were subjected to in vitro
differentiation conditions (see Lee et al., Nat. Biotechnol.
18:675-9, 2000) that led to differentiation of astrocytes and
neurons.
[0074] Using protocols for the selection of progenitor cells, we
were able to select inner ear progenitor cells that express a
variety of marker genes indicative of the developing inner ear. In
particular, we found after selection from embryoid body-derived
cells, cell populations that expressed genes indicative of the otic
placode, such as Pax2, BMP4, and BMP7 (Morsli et al., J. Neurosci.
18:3327-35, 1998; Groves and Bronner-Fraser, Development
127:3489-99, 2000). In addition, we found expression of marker
genes for the developing sensory epithelia (for example, Math1
(Bermingham et al., Science 284:1837-41, 1999), delta1, jagged1 and
jagged2 (Lanford et al., Nat. Genet. 21:289-92, 1999; Morrison et
al., Mech. Dev. 84:169-72, 1999)). Gene expression was detected by
reverse transcription followed by polymerase chain reaction
(RT-PCR). The differentiated cells were analyzed 14 days after the
removal of bFGF from the culture. The expression of the marker
genes correlated with the developmental stage of the progenitor or
mature cells as nestin and Pax2 and BMP7 expression decreased upon
differentiation of the cells and appearance of hair cell
markers.
[0075] Hair cell markers in differentiated cells were also detected
by immunohistochemistry. The hair cells produced in this system
co-expressed markers important for hair cell differentiation
(Math1) and survival (Brn3.1) and markers present in the more fully
differentiated cells (myosin VIIa).
[0076] In preliminary experiments, we explored whether it was
feasible to isolate clonal lines that represent hair cell and
neural progenitors from embryoid bodies. We were able to generate
spheres that contained progenitors, which we identified by
expression of the early neural marker Sox1 and the intermediate
filament protein nestin. We were able to propagate these progenitor
cells in serum-free conditions for more than three months either in
the form of spheres or as adherent cultures in the presence of
mitogenic growth factors. We routinely observed differentiation of
the progenitor cells after removal of growth factors in adherent
cultures.
Example 3
Different Neural Progenitor Cells were Generated from ES Cells
[0077] We determined that it was feasible to use embryoid bodies to
isolate clonal lines that represent neural progenitors. One goal of
the project was to generate neurons with different features that
could be used to generate neural populations having characteristics
similar to spiral ganglion neurons. The principal idea of this
technique was to use the sphere-forming capacity of neural stem
cells to clone different cell lines. Our initial results indicated
that we were able, for example, to generate spheres that contain
neural progenitors, which we identified by expression of the early
neural marker Sox1 and the intermediate filament protein
nestin.
[0078] We were able to propagate these neural progenitor cells in
serum-free conditions for more than three months either as spheres
or as adherent cultures in the presence of mitogenic growth
factors. We routinely observed neural differentiation of the
progenitor cells either in aging spheres or after removal of growth
factors in adherent cultures. In experiments done with Sox1-GFP ES
cells, we were able to generate proliferating neural progenitor
lines that expressed nestin and Sox1, visible in real-time by green
fluorescence. These cells readily differentiated into
morphologically and immunologically distinct neurons after removal
of mitogenic growth factors.
[0079] We examined the electrophysiological properties of neurons
generated from embryonic stem cells and from stem cells harvested
from adult ears. Using the strategy outlined above we examined
embryonic stem cells differentiated to become presumptive auditory
sensory neurons. The cells adopted neural morphology and acquired
negative resting potentials and the ability to fire action
potentials.
Example 4
Development of an Assay for Differentiation of ES Cells
[0080] In order to more systematically test the effects of
different genes or compounds on the conversion of ES cells to cells
resembling spiral ganglion neurons, we developed a luciferase assay
system in which the conversion of the progenitors to the desired
cell types is readily detected by a reporter construct. The aim was
to have the reporter construct under the control of a promoter that
is activated in the differentiated cell but is inactive in the
progenitor cells, so that a luciferase signal is generated by
differentiation of the cells. The assay can be performed using
conditions known to be useful for generating neurons from ES cells.
Cells that are grown in the presence of growth factors are cultured
in medium without growth factors, and this induces their
differentiation to neurons based on the expression of markers.
Under these conditions, the reporter cells will differentiate and
generate a signal. We used mouse ES cells (ROSA 26) to generate
neural progenitors in the presence of EGF, IGF-1 and bFGF. The
neural progenitors were used for construction of the reporter cell
lines. The progenitor cells were positive for nestin expression and
were kept in culture in the presence of bFGF.
[0081] To determine whether a cell-specific promoter could be
measured in this assay, neural progenitors were co-transfected with
the firefly luciferase gene controlled by a GFAP promoter and a
vector that contains the Renilla luciferase gene under control of a
CMV promoter. The firefly luciferase construct was made in the pGL3
basic vector (Promega, Madison, Wis.) that contains the firefly
luciferase gene and a multiple cloning site for the promoter. The
GFAP promoter inserted into this site allowed us to measure the
activity of this promoter relative to the constitutively active
control promoter in a separate vector driving the Renilla
luciferase. Co-transfection of the vectors into the neural
progenitors followed by lysis of the cells and measurement of
luciferase activity (using two substrates for measurement of
firefly and Renilla luciferase) allowed us to demonstrate that the
neural progenitors were initially negative for GFAP expression but
after removing bFGF from the culture medium, had increasing amounts
of luciferase activity (at 24, 48 and 72 hours). Furthermore, the
neural progenitor cells expressed the Renilla and firefly
luciferases at levels that were proportional to the amount of
vector used for transfection. These results indicate that the assay
is useful for determining quantitative effects relating to the
differentiation of the cells in response to individual genes or
factors.
Example 5
ES Cell-Derived Progenitors were Grafted into a Developing Chicken
Inner Ear
[0082] We established microsurgical techniques to manipulate
developing chicken ears. For injection of ES cell-derived
progenitors, we used beveled glass-capillary micropipettes for
injections into the otic pits or vesicles of stage 10-16 chicken
embryos (1.5-2.5 days of embryonic development, (Hamburger and
Hamilton, J. Morphol. 88:49-92, 1951)). Genetically labeled ES
cell-derived inner ear progenitors were implanted into the inner
ear of chicken embryos and their fate was followed through early
otic development. The cells were observed to be engrafted into a
preexisting epithelium and certain criteria were identified as
being necessary for the cells to engraft. Progenitor cells only
survived when implanted as cell aggregates. Progenitor cells that
were injected into the otic vesicle in the form of suspensions were
not traceable. Integration of cells from the progenitor cell
aggregates into the epithelial layers that form the otic vesicle
occurred preferentially at sites of epithelial damage. The
progenitor-derived cells were incorporated throughout the inner
ear, but in our study, we only focused on hair cell development.
Murine cells only upregulated hair cell markers when situated in a
developing sensory epithelium and only when they were located on
the luminal site of the epithelium--in the correct orientation for
hair cells. Progenitor-derived cells that we found elsewhere in the
inner ear did not display expression of hair cell markers.
[0083] In addition to the repopulation of the sensory epithelium
(Li et al., Proc. Natl. Acad. Sci. USA 100:13495-500, 2003), we
also found progenitor cell derivatives outside of the sensory
epithelia in the auditory ganglion. In fact, we initially observed
more efficient integration of cells into the auditory ganglion than
into the cochlear sensory epithelium.
Example 6
Transplantation-Repair Studies in the De-Afferented Cat
[0084] A unilaterally de-afferented cat is a useful animal model
for the study of sensorineural hearing loss with either primary
neural degeneration or primary hair cell damage followed by
secondary neural degeneration. We cut the auditory nerve in cats
and allowed them to survive for up to 1 year post surgery. Such
surgery can result in near complete loss of the auditory nerve, yet
all other structures of the cochlea remain normal. Months after
nerve section, there appeared to be a reinnervation of the organ of
Corti by branches of the facial nerve, which can be seen, in serial
sections, streaming through the ganglion without a soma. Within the
organ of Corti, this reinnervation appeared as spiraling fibers
lining either side of the inner hair cell. These results suggested
(i) that hair cells can survive in the adult ear without their
afferent innervation, and (ii) that the surviving hair cells are
likely expressing signals that remain capable of attracting new
neural contacts.
[0085] This animal model was used as a platform for neural
transplantation studies. As shown in FIGS. 3A and 3B, the
distortion product otoacoustic emissions (DPOAEs) remained normal
in the de-afferented ear, while there was a dramatic elevation of
compound action potential (CAP) thresholds in the de-afferented
ear. These results indicated that all the processes underlying
transduction and amplification in the cochlea were normal in the
de-afferented ear. Therefore, this model system is ideally suited
to a neural transplantation experiment.
[0086] We have performed a number of xenotransplantation
experiments in the unilaterally de-afferented cat and assessed the
extent of incorporation and differentiation of progenitor cells up
to 8 weeks post transplantation. The basic approach in the eight
animals studied to date has been to (1) cut the auditory nerve
bundle near the Schwann glial border, (2) put the animals on
cyclosporin immunosuppression therapy, (3) inject neural progenitor
cells after a variable recovery interval from 0 to 12 weeks, (4)
allow a post-implantation survival of 1-8 weeks, (5) assess
functional recovery via a terminal electrophysiological session,
and (6) harvest the cochlea and the brain for histological
verification of the extent of the primary neural degeneration and
the survival and differentiation of transplanted cells.
[0087] The progenitor cells injected have included (i) immature
spiral ganglion neurons isolated from fetal pigs and (ii) mouse ES
cells, each expressing .beta.-galactosidase reporters. In some
cases, the exogenous cells were transplanted into the round window
and in other cases into the auditory nerve, just peripheral to the
site of the surgical section.
[0088] In one study, ES cells were transplanted into the auditory
nerve four weeks after surgery. Functional recovery was assessed
via a terminal electrophysiological session, and when the animal
was sacrificed six weeks after transplantation,
.beta.-galactosidase positive cells were seen only in the vicinity
of the electrode track (none were seen anywhere else in the nerve
or cochlear nucleus). Some of these cells had neural morphology. In
one case, a total of 150 .beta.-gal positive cells were seen near
the electrode track.
Other Embodiments
[0089] A number of embodiments of the invention have been
described. Nevertheless, it will be understood that various
modifications may be made without departing from the spirit and
scope of the invention. Accordingly, other embodiments are within
the scope of the following claims.
* * * * *